Method for determining the antimicrobial agent sensitivity of a nonparaffinophilic hydrophobic microorganism

ABSTRACT

A method for determining a sensitivity of a nonparaffinophilic hydrophobic microorganism to an antimicrobial agent. The method includes providing at least one receptacle containing an aqueous broth including a carbon source and introducing the nonparaffinophilic hydrophobic microorganism into the receptacle. The method further includes placing into the receptacle (i) a slide coated with a hydrophobic material and (ii) a predetermined quantity of the antimicrobial agent to be tested. By observing the nonparaffinophilic hydrophobic microorganism growth or lack thereof on the slide, it can be determined whether the predetermined quantity of the antimicrobial agent is effective in inhibiting growth of the nonparaffinophilic hydrophobic microorganism on the slide. An associated apparatus is also disclosed.

BACKGROUND OF THE INVENTION

This invention relates to a method for determining the antimicrobialagent sensitivity of a nonparaffinophilic hydrophobic microorganism andan associated apparatus.

Treating infections very often involves educated guesses by medicalpersonnel as to the nature of the microorganism involved and the correctantimicrobial agent and quantity thereof needed to effectively treat themicroorganism present in the infected tissue. Medical personnel areacutely interested in rapidly ascertaining which antimicrobial agents,and which dosages, are necessary in order to assure effective inhibitionof the growth of all microorganisms present in the patient.

Several of my United States Patents teach an effective, efficient andeconomical way for medical personnel to rapidly ascertain whichantimicrobial agent and which dosage is necessary in order to treat thepatient. See U.S. Pat. No. 5,663,056, the disclosure of which isincorporated by reference herein. Despite the existence of thistechnology, there still remains a need to determine the antimicrobialagent sensitivity of nonparaffinophilic hydrophobic microorganisms.

SUMMARY OF THE INVENTION

The invention has met or exceeded the above-mentioned need as well asothers. The method for determining the sensitivity of hydrophobicnonparaffinophilic microorganism to an antimicrobial agent and comprisesproviding at least one receptacle containing an aqueous broth includinga carbon source and introducing into the receptacle thenonparaffinophilic hydrophobic microorganism. The method furtherincludes placing into the receptacle (i) a slide coated with ahydrophobic material and (ii) a predetermined quantity of theantimicrobial agent to be tested. By observing the nonparaffinophilichydrophobic microorganism growth or lack thereof on the slide, it can bedetermined whether the predetermined quantity of the antimicrobial agentis effective in inhibiting growth of the nonparaffinophilic hydrophobicmicroorganism on the slide.

An associated apparatus is also disclosed. The apparatus comprises areceptacle adapted to contain an aqueous broth including a carbonsource, an amount of antimicrobial agent to be tested and thenonparaffinophilic hydrophobic microorganism. The apparatus furtherincludes a slide coated with a hydrophobic material, the slide beingadapted to being placed in the receptacle. Again, observation of thegrowth of the nonparaffinophilic hydrophobic microorganism on the slidecan be used to determine the concentration of the antimicrobial agentnecessary to resist the nonparaffinophilic hydrophobic microorganismgrowth on the slide.

BRIEF DESCRIPTION OF THE DRAWING

A full understanding of the invention can be gained from the followingdetailed description of the invention when read in conjunction with theaccompanying lone drawing which shows one embodiment of theantimicrobial agent sensitivity apparatus.

DETAILED DESCRIPTION

As used herein, the term "nonparaffinophilic hydrophobic microorganism"means a nonparaffinophilic microorganism that, when inoculated into anaqueous broth containing a dissolved carbon source, will prefer to growupon a hydrophobic surface introduced into the broth as opposed togrowing within the aqueous broth itself. Examples of suchnonparaffinophilic hydrophobic microorganisms include, but are notlimited to, the following: M. tuberculosis complex (M. tuberculosis, M.bovis, M. africanum, M. microti); M. paratuberculosis; M. leprae;pseudomonads; and nocardial species.

As used herein, the term "nonparaffinophilic microorganism" means anymicroorganism sustained by a carbon source other than paraffin. Also, asused herein, the term "patient" refers to a member of the animalkingdom, including human beings

The method and apparatus of the invention provide an efficient,effective and economical way of determining the sensitivity of anonparaffinophilic hydrophobic microorganism to different antimicrobialagents and predetermined quantities thereof. Referring now to the loneFIGURE, the antimicrobial agent sensitivity method will be explainedwith reference to one embodiment of the antimicrobial agent sensitivityapparatus 10. The apparatus 10 consists of six receptacles in the formof test tubes 20, 21, 22, 23, 24, 25 each containing an amount of anaqueous broth including a carbon source, such as Middlebrook 7H9 broth,40, 41, 42, 43, 44, 45. The aqueous broth in test tubes 21-25 containuniform intervals of increasing concentrations of an antimicrobial agentto be tested. Test tube 20 is used as a control tube that does notcontain any antimicrobial agent.

The nonparaffinophilic hydrophobic microorganism isolate is thenintroduced into each of the test tubes 20-25. This isolate can beobtained from a patient's body specimen. The body specimen can besputum, fecal matter, cerebrospinal fluid, urine, gastric fluid,lymphatic material and purulent body fluids. These specimens can beobtained from the patient in the doctor's office or in the emergencyroom of a hospital, for example, by known techniques.

Slides 50, 51, 52, 53, 54 and 55 are coated with a hydrophobic material(defined further below) 60, 61, 62, 63, 64 and 65 (shown in partialcutaway sections on each slide), respectively, are then placed intorespective test tubes 20, 21, 22, 23, 24 and 25. The slides areincubated for a period of a minimum of twenty-four (24) hours andpossibly from 5 to 20 days. By observing nonparaffinophilic hydrophobicmicroorganism growth 90, 91, 92, 93, 94 on the slides 50-54, the minimuminhibitory concentration ("MIC") of the antimicrobial agent necessary toprevent nonparaffinophilic hydrophobic microorganism growth can bedetermined. In this case, hydrophobic material 65 coated on slide 55 hasno nonparaffinophilic hydrophobic microorganism growth, thus the MICconcentration is found in test tube 55.

It will be appreciated that although apparatus 10 is shown with multiplereceptacles and multiple slides 50-55, that the invention is not limitedto multiple receptacles and multiple slides, but covers also a singlereceptacle and a single slide.

The hydrophobic materials that can be used are paraffin wax and otherwaxes; plastics such as polypropylene, polyethylene, polystyrene andtetrafluoroethylene; or silicones.

The invention utilizes the inventor's unexpected finding that M.tuberculosis and M. paratuberculosis and other microorganisms were ableto grow on paraffin wax or other hydrophobic material in a broth mediacontaining a dissolved carbon source, such as Middlebrook 7H9 broth. Thewaxy cell walls of these microorganisms, it is thought, favorsattachment to a hydrophobic surface, such as paraffin wax, and thus themicroorganism can imbibe the nutrients in the broth while being attachedto the paraffin wax. This so-called "hydrophobic baiting" can be used todetermine the antimicrobial agent sensitivity of thesenonparaffinophilic hydrophobic microorganisms.

EXAMPLE

A patient is determined to have M. tuberculosis and the doctor wants todetermine the appropriate dosage of the antimicrobial agentstreptomycin. The M. tuberculosis isolate from the body specimen of thepatient is introduced into receptacles 20-25 containing (1) the aqueousMiddlebrook 7H9 broth and (2) the slide 50-55 containing the hydrophobiccoating material. The following amounts of streptomycin are introducedinto each receptacle.

    ______________________________________    Receptacle   Amount    ______________________________________    20           0            (Control)    21           100          μg/ml    22           325          μg/ml    23           550          μg/ml    24           775          μg/ml    25           1000         μg/ml    ______________________________________

After an incubation period of between 7-60 days, there is growth on allslides except slide 25. Therefore, the MIC of the streptomycin isdetermined to be 1000 μg/ml.

It will be appreciated that a method and kit are provided to determinethe antimicrobial agent sensitivity of a nonparaffinophilic hydrophobicmicroorganism. The method is simple and efficient and the kit itself isfairly inexpensive.

While specific embodiments of the invention have been disclosed, it willbe appreciated by those skilled in the art that various modificationsand alterations to those details could be developed in light of theoverall teachings of the disclosure. Accordingly, the particulararrangements disclosed are meant to be illustrative only and notlimiting as to the scope of the invention which is to be given the fullbreadth of the appended claims and any and all equivalents thereof.

What is claimed is:
 1. A method for determining a sensitivity of anonparaffinophilic hydrophobic microorganism to an antimicrobial agent,said method comprising:providing at least one receptacle containing anaqueous broth including a carbon source; introducing into saidreceptacle said nonparaffinophilic hydrophobic microorganism; placinginto said receptacle (i) a slide coated with a hydrophobic materialselected from the croup consisting of waxes, silicones and plastics and(ii) a predetermined quantity of said antimicrobial agent; and observingsaid nonparaffinophilic hydrophobic microorganism growth or lack thereofon said slide to determine whether said predetermined quantity of saidantimicrobial agent is effective in inhibiting growth of saidnonparaffinophilic hydrophobic microorganism on said slide.
 2. Themethod of claim 1, whereinsaid nonparaffinophilic hydrophobicmicroorganism is selected from the group consisting of M. tuberculosiscomplex, M. paratuberculosis, M. leprae, pseudomonads, and nocardialspecies.
 3. The method of claim 1, whereinsaid hydrophobic material isparaffin wax.
 4. The method of claim 1, includingproviding a pluralityof receptacles each containing an aqueous broth including a carbonsource; introducing into each receptacle said nonparaffinophilichydrophobic microorganism; placing (i) a separate slide coated with ahydrophobic material selected from the group consisting of waxes,silicones and plastics and (ii) an amount of an antimicrobial agent tobe tested in each said receptacle, each said receptacle containing adifferent predetermined quantity of said antimicrobial agent; andobserving said nonparaffinophilic hydrophobic microorganism growth orlack thereof on said slides to determine the minimum inhibitoryconcentration of said antimicrobial agent to inhibit growth of saidnonparaffinophilic hydrophobic microorganism.